The Alpha-ketoacid-dependent dioxygenases are a class of enzymes which catalyze the hydroxylation of aliphatic and aromatic carbon atoms in a variety of substrates. These iron containing proteins are vital to amino acid degradation, the biosynthesis of carnitine, and the post-translational modification of collagen, but their mechanisms of catalysis are poorly understood. We will use modified substrates of these enzymes as probes of their modes of action. Specifically, we will: 1) Purify two representative examples of these proteins -- p-hydroxyphenylpyruvate dioxygenase (which catalyzes the second reaction of tyrosine catabolism) and Gamma-butyrobetaine dioxygenase (which catalyzes the last step in carnitine biosynthesis) -- by literature procedures. 2) Synthesize isotopically-labeled substrates and potential alternate substrates containing mechanistically informative reactive functionality at key sites of the molecules. 3) Incubate these compounds with the enzymes, determine their competence as substrates and/or inhibitors, and determine the structures of the incubation products in order to address the following mechanistic points of general importance to the understanding of all Alpha-ketoacid dioxygenases: a) The nature of the highly reactive carbon oxygenating agent. b) The stereochemical course of oxygenation. c) The mechanism and function of Alpha-ketoacid oxidative decarboxylation. d) The possible presence and role of radical and carbanionic intermediates in catalysis. Evaluation of the results from these experiments will aid in forming a conclusion about the nature and sequence of events at the active sites of these important enzymes during catalysis.